This new proposal focuses on cross-disciplinary characterizations of protein metal ion based biosensors. These biosensors include key virulence factors that act in pathogenesis. All these proteins influence cellular metabolism through the metal-mediated control of gene expression by nucleic acid interactions. This function of these protein biosensors involves metal-induced protein conformational changes expected to be large and suitable for elucidation by the proposed crystallography characterizations. These structural efforts will be synergistically coupled with biochemistry, genetics, and spectroscopy results. Structural and mutational studies of the bacterial transcriptional regulators ferric uptake regulator (Fur) and the zinc uptake regulator (Zur) will reveal how metal ion binding is structurally linked to rearrangements of a DNA binding surface. Importantly, metal binding provides sufficient binding energy to drive protein conformational changes yet maintain sufficient affinity so that these changes are triggered at physiologically relevant concentrations for the control of bacterial pathogenesis. The superoxide stress regulator (SoxR) acts as a redox sensor to drive pathogen responses to oxidative stress induced by the environment and the mammalian immune response. Conformational changes that convert SoxR from a repressor to an activator of transcription are induced via the single electron oxidation of a [2Fe-2S] cluster. SoxR crystal structures in apo and in metal-bound, defined oxidation states will be combined with site directed mutants and density functional theory calculations of changes in partial charge distribution to delineate the mechanism by which this information is transduced through the protein architecture. The mammalian iron regulatory protein (IRP1) uses the assembly of a [4Fe-4S] cluster as an iron sensor to regulate the translation of genes involved in iron metabolism. Crystal structures of apo IRP in complex with its RNA binding target will reveal the means whereby the protein can stabilize two discrete conformations. Together, these aims will address mechanisms by which metal ion switches are coupled to protein conformational changes that allow pathogens and mammalian to sense small molecule ligands, redox states, and metal concentration within the cell. [unreadable] [unreadable] [unreadable]